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STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Grant No.
EFRI-0937997 awarded by the National Science Foundation. The Government
has certain rights in this invention.

Claims

1. A sensor comprising: a substrate; at least one electrode contacting
the substrate; a nanoporous membrane covering the electrode; and a
biorecognition element selected from the group consisting of a peptide,
an antibody, an enzyme, and an aptamer, wherein the biorecognition
element is covalently bound to the electrode, or covalently bound to a
PEDOT random copolymer embedded within the nanoporous membrane, the PEDOT
random copolymer having a structure according to Formula I: ##STR00012##
wherein each R is independently selected from the group consisting of
--OH and the biorecognition element, wherein at least one R is the
biorecognition element, and x and y are independently an integer of from
about 1 to about 1000, wherein the sum of x and y is an integer of from
about 2 to about 1000.

2. The sensor of claim 1, wherein the electrode is a micropatterned gold
electrode.

3. The sensor of claim 1, wherein the sensor comprises a center working
electrode, a surrounding counter electrode, and a reference electrode.

4. The sensor of claim 1, wherein the biorecognition element is an
antibody.

5. The sensor of claim 1, wherein the biorecognition element is an
aptamer.

6. The sensor of claim 1, wherein the biorecognition element is
covalently bound to the PEDOT random copolymer embedded within the
nanoporous membrane, wherein the nanoporous membrane comprises a PEG
hydrogel having PEG chains with a molecular weight of from about 1000 Da
to about 10,000 Da.

7. The sensor of claim 6, wherein the PEG hydrogel comprises PEG chains
having a molecular weight of about 6000 Da.

8. The sensor of claim 6, wherein the PEG hydrogel is covalently bound to
the substrate.

9. The sensor of claim 6, wherein the ratio of x to y is from about 10:1
to about 1:10.

10. The sensor of claim 1, wherein the nanoporous membrane is an aluminum
oxide membrane.

11. The sensor of claim 10, wherein the biorecognition element is
covalently linked to the electrode to form a self-assembled monolayer of
the biorecognition element on the electrode.

12. The sensor of claim 11, wherein the biorecognition element is an
aptamer having a redox reporting moiety and a thiol moiety, wherein the
thiol moiety is covalently bound to a gold working electrode.

13. A method for detecting a disease marker in a biological sample
comprising contacting a sensor according to claim 1 with the biological
sample and detecting the binding of the disease marker to a
biorecognition element, thereby detecting the disease marker.

14. The method of claim 13, wherein detecting the binding of the disease
marker to the biorecognition element comprises measuring the peak
reduction current of the PEDOT random copolymer.

15. The method of claim 13, wherein the binding of the disease marker to
the biorecognition element is detected using square wave voltammetry.

16. The method of claim 13, wherein the disease marker is indicative of
an infection by tuberculosis or hepatitis C.

17. A conductive hydrogel comprising: a covalently cross-linked
poly(ethylene glycol) (PEG) hydrogel; and a
poly(3,4-ethylenedioxythiophene) (PEDOT) random copolymer embedded within
the PEG hydrogel, the PEDOT random copolymer having a structure according
to Formula I: ##STR00013## wherein each R is independently selected
from the group consisting of --OH and a biorecognition element selected
from the group consisting of a peptide, an antibody, an enzyme, and an
aptamer, wherein at least one R is the biorecognition element, and x and
y are independently an integer of from about 1 to about 1000, wherein the
sum of x and y is an integer of from about 2 to about 1000.

18. The conductive hydrogel of claim 17, wherein the PEG hydrogel
comprises PEG chains having molecular weights of from about 1000 Da to
about 10,000 Da.

19. The conductive hydrogel of claim 17, wherein the ratio of x to y is
from about 10:1 to about 1:10.

[0001] The present application is a continuation of PCT/US2014/071596,
filed Dec. 19, 2014, which application claims the benefit of priority to
U.S. Provisional Patent Application No. 61/918,099 filed Dec. 19, 2013,
all of which applications are incorporated herein by reference in their
entireties.

BACKGROUND OF THE INVENTION

[0003] Conducting polymers hold significant promise in biomolecular
electronics as electrode coatings and provide high electrical
conductivity. However, they are characterized by inherently poor
mechanical properties. Incorporation of hydrogels with conducting
polymers may act to modulate and improve mechanical properties, as well
as provide a non-fouling surface and a depot for water soluble, bioactive
agents. Besides this, blends of electro-active polymer and biocompatible
hydrogel provide three-dimensional constructs for greater biomolecule
immobilization with enhanced sensitivity and hence are promising
materials for biosensor development.

[0004] Among all known conducting polymers,
poly(3,4-ethylenedioxythiophene) (PEDOT) exhibits very low intrinsic
cytotoxicity and display no inflammatory response upon implantation,
making them ideal for biosensing and bioengineering applications.
Polyethylene glycol (PEG) is a biocompatible polymer known for its
excellent non-fouling properties. PEG has been used widely to minimize
unwanted protein adsorption and cell attachment in tissue engineering and
biosensor development. We believe that the amalgam of properties of PEDOT
and PEG may provide promising biosensing materials.

BRIEF SUMMARY OF THE INVENTION

[0005] In one embodiment, the present invention provides a sensor having a
substrate; at least one electrode contacting the substrate; a nanoporous
membrane covering the electrode; and a biorecognition element selected
from the group consisting of a peptide, an antibody, an enzyme, and an
aptamer, wherein the biorecognition element is covalently bound to the
electrode, or covalently bound to a PEDOT random copolymer embedded
within the nanoporous membrane, the PEDOT random copolymer having a
structure according to Formula I:

##STR00001##

wherein each R is independently selected from the group consisting of
--OH and the biorecognition element, wherein at least one R is the
biorecognition element, and x and y are independently an integer of from
about 1 to about 1000, wherein the sum of x and y is an integer of from
about 2 to about 1000.

[0006] In another embodiment, the present invention provides a method for
detecting a disease marker in a biological sample comprising contacting a
sensor of the present invention with the biological sample and detecting
the binding of the disease marker to a biorecognition element, thereby
detecting the disease marker.

[0007] In another embodiment, the present invention provides a conductive
hydrogel comprising a covalently cross-linked poly(ethylene glycol) (PEG)
hydrogel; and a poly(3,4-ethylenedioxythiophene) (PEDOT) random copolymer
embedded within the PEG hydrogel, the PEDOT random copolymer having a
structure according to Formula I:

##STR00002##

wherein each R is independently selected from the group consisting of
--OH and a biorecognition element selected from the group consisting of a
peptide, an antibody, an enzyme, and an aptamer, wherein at least one R
is the biorecognition element, and x and y are independently an integer
of from about 1 to about 1000, wherein the sum of x and y is an integer
of from about 2 to about 1000.

[0008] In another embodiment, the present invention provides a method for
preparing the conductive polymer of the present invention, comprising
contacting a PEG-diacrylate and a photoinitiator under polymerization
conditions suitable to form a PEG hydrogel; contacting the PEG hydrogel
with a solution comprising 3,4-ethylenedioxythiophene (EDOT) and
2,3-dihydrothieno[3,4-b][1,4]dioxine-2-carboxylic acid (EDOT-COOH) under
electropolymerization conditions sufficient to form a
poly(3,4-ethylenedioxythiophene) (PEDOT) random copolymer of Formula I
embedded within the PEG hydrogel:

##STR00003##

wherein each R is --OH, and x and y are each an integer of from about 1
to about 1000, wherein the sum of x and y is an integer of from about 2
to about 1000; and contacting the hydrogel with a biorecognition element
under conditions sufficient to covalently bind the biorecognition element
to the PEDOT random copolymer, thereby forming the PEDOT random copolymer
of Formula I wherein at least one R is a biorecognition element selected
from the group consisting of a peptide, an antibody, and an aptamer,
thereby preparing the conductive hydrogel of the present invention.

[0026] FIG. 17 shows a schematic drawing of the sensor of the present
invention, including the location of the reference, working and counter
electrodes.

[0027] FIG. 18A and FIG. 18B show various embodiments of the biosensor
(100) of the present invention in cross-section, including a substrate
(110), electrodes (120), a nanoporous membrane (130) with biorecognition
elements (140), and a self-assembled monolayer (150) on the substrate.

[0028] FIG. 19A and FIG. 19B shows various embodiments of the biosensor
(200) of the present invention in cross-section, including a substrate
(210), electrodes (220), a nanoporous membrane (230), with biorecognition
elements (240) linked to the electrodes via a self-assembled monolayer
(250) on the electrodes.

DETAILED DESCRIPTION OF THE INVENTION

I. General

[0029] The present invention provides a sensor for detection of biological
agents and detecting of disease. The sensor includes a biorecognition
element attached to a conducting polymer that covers an electrode, so
that binding of the biorecognition element to the biological agent
results in a change in the measured potential for the electrode, thus
detecting the binding event. The sensor enables detection without the use
of a label. Moreover, the conducting polymer, a PEDOT-PEG hydrogel, has
improved mechanical properties and better resists delamination from the
electrode due to covalent binding to the substrate surface.

II. Definitions

[0030] "Substrate" refers to any solid material suitable for supporting
the sensor of the present invention. Suitable substrates can include
materials such as silicon, silicon dioxide and indium-tin-oxide. Examples
of suitable substrates include, but are not limited to, glass (including
controlled-pore glass), polymers (e.g., polystyrene, polyurethane,
polystyrene-divinylbenzene copolymer), silicone rubber, quartz, latex, a
transition metal, magnetic materials, silicon dioxide, silicon nitride,
gallium arsenide, and derivatives thereof. Except for the reactive sites
on the surface, substrate materials are generally resistant to the
variety of chemical reaction conditions to which they may be subjected.
The substrate useful in the present invention can be smooth or roughened.
A smooth surface is one having a minimum of features on the surface that
lead to roughness. A roughened surface is one that has a multitude of
features on the surface that create friction. The substrate of the
present invention includes a first substrate and a second substrate. The
substrate can be flat or non-flat, flexible or rigid. In addition, the
substrate can be porous, mesh or fabric. The substrate can be opaque or
transparent, and can transmit or reflect light, or both. One of skill in
the art will appreciate that other substrates are useful in the present
invention.

[0031] "Electrode" refers to an electrical conductor for making contact
with a part of a circuit. A circuit can often include several electrodes
working together, such as the working electrode, the counter electrode
and the reference electrode. The working electrode is the electrode in
the system where the reaction or interaction of interest takes place. The
counter electrode assists the working electrode in the measurement taking
place. The reference electrode provides a stable electrode potential
against which the potential of the working and counter electrodes are
measured.

[0032] "Membrane" refers to a layer that allows material and fluids to
move from one location to another through the membrane. The membrane can
prevent some material from moving due to size limitations of the pores.
When the pores are nanometer sized, the membrane can be a nanoporous
membrane.

[0033] "Contacting" refers to bringing into close proximity at least two
distinct objects such that a portion of at least one surface of the first
object comes into contact with a portion of at least one surface of the
second object.

[0034] "Covalently bound" refers to the formation of a covalent bond
between the substrate of the present invention and a portion of the
hydrogel.

[0035] "Biorecognition element" refers to a biological agent capable of
specifically binding to another biological agent.

[0036] "Polypeptide," "peptide," and "protein" are used interchangeably
herein to refer to a polymer of amino acid residues. All three terms
apply to amino acid polymers in which one or more amino acid residue is
an artificial chemical mimetic of a corresponding naturally occurring
amino acid, as well as to naturally occurring amino acid polymers and
non-naturally occurring amino acid polymers. As used herein, the terms
encompass amino acid chains of any length, including full-length
proteins, wherein the amino acid residues are linked by covalent peptide
bonds.

[0038] An antibody immunologically reactive with a particular antigen can
be generated by recombinant methods such as selection of libraries of
recombinant antibodies in phage or similar vectors, see, e.g., Huse et
al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546
(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or by
immunizing an animal with the antigen or with DNA encoding the antigen.

[0039] Typically, an immunoglobulin has a heavy and light chain. Each
heavy and light chain contains a constant region and a variable region,
(the regions are also known as "domains"). Light and heavy chain variable
regions contain four "framework" regions interrupted by three
hypervariable regions, also called "complementarity-determining regions"
or "CDRs". The extent of the framework regions and CDRs have been
defined. The sequences of the framework regions of different light or
heavy chains are relatively conserved within a species. The framework
region of an antibody, that is the combined framework regions of the
constituent light and heavy chains, serves to position and align the CDRs
in three dimensional space.

[0040] The CDRs are primarily responsible for binding to an epitope of an
antigen. The CDRs of each chain are typically referred to as CDR1, CDR2,
and CDR3, numbered sequentially starting from the N-terminus, and are
also typically identified by the chain in which the particular CDR is
located. Thus, a V.sub.H CDR3 is located in the variable domain of the
heavy chain of the antibody in which it is found, whereas a V.sub.L CDR1
is the CDR1 from the variable domain of the light chain of the antibody
in which it is found.

[0041] References to "V.sub.H" or a "VH" refer to the variable region of
an immunoglobulin heavy chain of an antibody, including the heavy chain
of an Fv, scFv, or Fab. References to "V.sub.L" or a "VL" refer to the
variable region of an immunoglobulin light chain, including the light
chain of an Fv, scFv, dsFv or Fab.

[0042] The phrase "single chain Fv" or "scFv" refers to an antibody in
which the variable domains of the heavy chain and of the light chain of a
traditional two chain antibody have been joined to form one chain.
Typically, a linker peptide is inserted between the two chains to allow
for proper folding and creation of an active binding site.

[0043] A "chimeric antibody" is an immunoglobulin molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is linked to
a constant region of a different or altered class, effector function
and/or species, or an entirely different molecule which confers new
properties to the chimeric antibody, e.g., an enzyme, toxin, hormone,
growth factor, drug, etc.; or (b) the variable region, or a portion
thereof, is altered, replaced or exchanged with a variable region having
a different or altered antigen specificity.

[0044] A "humanized antibody" is an immunoglobulin molecule which contains
minimal sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in which
residues from a complementary determining region (CDR) of the recipient
are replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired specificity,
affinity and capacity. In some instances, Fv framework residues of the
human immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found neither
in the recipient antibody nor in the imported CDR or framework sequences.
In general, a humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the framework (FR) regions
are those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et
al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992)). Humanization can be essentially performed following
the method of Winter and co-workers (Jones et al., Nature 321:522-525
(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat.
No. 4,816,567), wherein substantially less than an intact human variable
domain has been substituted by the corresponding sequence from a
non-human species.

[0045] "Epitope" or "antigenic determinant" refers to a site on an antigen
to which an antibody binds. Epitopes can be formed both from contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary folding
of a protein. Epitopes formed from contiguous amino acids are typically
retained on exposure to denaturing solvents whereas epitopes formed by
tertiary folding are typically lost on treatment with denaturing
solvents. An epitope typically includes at least 3, and more usually, at
least 5 or 8-10 amino acids in a unique spatial conformation. Methods of
determining spatial conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,
Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn
E. Morris, Ed (1996).

[0046] "Enzyme" refers to a biological catalyst.

[0047] "Aptamer" refers to oligonucleic acids or peptides that can bind to
a specific target molecule.

[0050] "PEDOT random copolymer" refers to a copolymer of
poly(3,4-ethylenedioxythiophene) (PEDOT) where EDOT and EDOT-COOH are
copolymerized together to form PEDOT having EDOT-COOH randomly
distributed in the copolymer:

##STR00006##

[0051] "Self-assembled monolayer" refers to a single layer of a substance
such as an organic compound, assembled on the surface of a substrate.
Self-assembled monolayers can be formed from a variety of materials, such
as silanes on silicon or thiols on gold.

[0052] "Redox reporting moiety" refers to any moiety capable of generating
a measurable signal in response to a change in the electrical potential.
The measurable change in the electrical current can be correlated to
concentration of the target analyte.

[0053] "Thiol moiety" refers to the group "--SH".

[0054] "PEG" refers to polyethylene glycol and polyethylene oxide
polymers. PEG can have any suitable molecular weight from less than 1,000
Daltons to over 100,000 Daltons. PEG can also be functionalized with a
variety of groups at either the alpha or omega end. For example, PEG can
be functionalized with one or more acrylate groups. When PEG is
functionalized with two acrylate groups, PEG-diacrylate is formed. PEG
can form a hydrogel by the crosslinking of PEG chains and the
incorporation of water into the crosslinked polymer matrix.

[0055] As used herein, the term "hydrogel" refers to a
highly-interdependent and interconnected, biphasic matrix having a solid
component (usually a polymer, and more commonly a highly cross-linked
polymer) that has both hydrophilic and hydrophobic character.
Additionally, the matrix has a liquid component (e.g., water) that is
retained in the matrix by intermolecular forces. The hydrophobic
character provides the matrix with a degree of water insolubility while
the hydrophilic character affords water permeability. One of skill in the
art will appreciate that several different types of polymers can be used
in combination to form hydrogels useful in the methods of the present
invention.

[0056] "Microtiter well plate" refers to a plate having a plurality of
wells for testing purposes, where the electrode is at the bottom of each
well. Each electrode can be surrounded by a well perimeter defining walls
around the electrode. Each well can be covered by a well cover.

[0057] "Disease marker" refers to a measurable indicator of the presence
or relative risk of a particular disease. The disease marker can be
genetic, or indicated by the relative concentration of a component in the
patient's blood or body relative to the normal concentration. The disease
marker can also be the relative function of a particular organ or process
in the body of the patient, relative to the normal function of the organ
or process.

[0058] "Biological sample" refers to any sample of the patient's body,
including tissue, organ, fluid, etc.

[0059] "Peak reduction current" refers to the current at the formal
potential of an electrochemical species that is being reduced.

[0060] "Square wave voltammetry" refers to the wave form employed in
electrochemistry whereby potential (voltage) is swept not in a linear
fashion but in a saw-tooth manner. This potential sweep method minimizes
capacitive currents and is preferred for sensitive measurements of small
changes in current.

[0061] "Tuberculosis" refers to the disease caused by myobacteria such as
myobacterium tuberculosis.

[0062] "Hepatitis C" refers to the disease caused by the Hepatitis C
virus, a member of the family Flaviviridae.

[0063] "Interferon-gamma" and "IFN-.gamma." refer to a type II interferon
that binds the type II IFN receptor, interfering with viral infection of
host cells. IFN-.gamma. regulates a variety of biological functions, such
as antiviral responses, cell growth, immune response, and tumor
suppression, and may mediate a variety of human diseases.

[0064] "Photoinitiator" refers to a compound that initiates a
polymerization process after irradiation. The photoinitiator can generate
acid (a photo-acid generator or PAG) or a radical, among other initiating
species. The acid, radical, or other species, then initiates a
polymerization.

[0065] "Electropolymerization conditions" refers to a polymerization
process that is initiated electrically instead of chemically. The
conditions for electropolymerization include application of an electrical
potential to the pre-polymer mixture with sufficient current and/or
charge to initiate the polymerization. The electropolymerization
conditions can vary depending on the polymer being prepared.

[0066] "Embedded" refers to the intercalation of the conductive polymer in
the nanoporous membrane where the conductive polymer is not covalently
linked to the nanoporous membrane.

III. Conductive Biosensors

[0067] The present invention provides a biosensor for the detection of
biological agents that can indicate a particular disease.

[0068] In some embodiments, the present invention provides a sensor having
a substrate; at least one electrode contacting the substrate; a
nanoporous membrane covering the electrode; and a biorecognition element
selected from the group consisting of a peptide, an antibody, an enzyme,
and an aptamer, wherein the biorecognition element is covalently bound to
the electrode, or covalently bound to a PEDOT random copolymer embedded
within the nanoporous membrane, the PEDOT random copolymer having a
structure according to Formula I:

##STR00007##

wherein each R is independently selected from the group consisting of
--OH and the biorecognition element, wherein at least one R is the
biorecognition element, and x and y are independently an integer of from
about 1 to about 1000, wherein the sum of x and y is an integer of from
about 2 to about 1000.

[0069] FIG. 18A-18B shows various embodiments of a biosensor (100) of the
present invention in cross-section, including a substrate (110),
electrodes (120), a nanoporous membrane (130) with biorecognition
elements (140), and a self-assembled monolayer (150) on the substrate.
FIG. 19A-19B also shows various embodiments of the biosensor (200) of the
present invention in cross-section, including a substrate (210),
electrodes (220), a nanoporous membrane (230), with biorecognition
elements (240) linked to the electrodes via a self-assembled monolayer
(250) on the electrodes. In some embodiments, the biorecognition element
(240) can be linked directly to the electrode (220) without an
intervening self-assembled monolayer (see FIG. 19B).

[0070] The substrate of the present invention can be any substrate
suitable for supporting electrodes and binding to the PEG hydrogel.
Examples of suitable substrates can be glass, insulators, ceramics,
semi-conductors, metals, polymers, etc. Representative substrates include
glass. In some embodiments, the substrate can be glass.

[0071] The biosensor of the present invention can include any suitable
number and type of electrodes (120) contacting the substrate. For
example, the biosensor can include a working electrode, a counter
electrode and a reference electrode. The electrodes can be made of any
suitable electrically conducting material. For example, the electrodes
can be made of a metal such as, but not limited to, gold, silver,
platinum, copper, etc. Other materials for the electrodes include metal
salts such as silver chloride. In some embodiments, the biosensor
includes a working electrode, a counter electrode and a reference
electrode. In some embodiments, the electrodes are made of gold.

[0072] The electrodes of the present invention can be oriented in any
suitable format relative to one another. For example, the electrodes can
be circular with one electrode surrounded by another electrode. Moreover,
the electrodes can be of any suitable shape, such as square, circular, or
torus shaped.

[0073] In some embodiments, the electrode is a micropatterned gold
electrode. In some embodiments, the sensor comprises a center working
electrode, a surrounding counter electrode, and a reference electrode. In
some embodiments, the working electrode is a gold electrode. In some
embodiments, the reference electrode is a silver/silver chloride
electrode.

[0074] The biosensor of the present invention can also include a
nanoporous membrane covering the electrodes. The nanoporous membrane can
be prepared from any suitable material. For example, the nanoporous
membrane can be a polymer, an inorganic material such as a ceramic or
oxide such as aluminum oxide, or other similar materials. The nanoporous
membrane can be conductive or non-conductive. The nanoporous membrane can
have pores of any suitable size, such as from 10 nm to 100 am. The
nanoporous membrane can be of any suitable thickness, such as from 10 nm
to 10 mm. In some embodiments, the nanoporous membrane can be aluminum
oxide. In some embodiments, the biosensor of the present invention does
not include a nanoporous membrane.

[0075] The nanoporous membrane can also be a conductive hydrogel. Any
suitable conductive hydrogel can be used in the present invention. The
hydrogel can be prepared from any suitable material, such as
polyethyleneglycol (PEG). The PEG can be functionalized with
crosslinkable groups such as acrylates, methacrylates or other
polymerizable groups. The PEG can be any suitable molecular weight. For
example, the PEG can have a molecular weight of about 1 kDs, 2, 3, 4, 5,
6, 7, 8, 9 or 10 kDa. PEG can also have a molecular weight up to about
100 kDa. In some embodiments, the PEG can be a PEG-diacrylate with a
molecular weight of about 6 kDa.

[0076] In some embodiments, the PEG hydrogel can be covalently linked to
the substrate. For example, the substrate can be modified with a
self-assembled monolayer capable of covalently binding to the
PEG-diacrylate during the polymerization process for preparing the PEG
hydrogel. In some embodiments, the substrate is modified with a
self-assembled monolayer containing an acrylate functional group.
Covalent attachment of the nanoporous membrane to the substrate can
reduce delamination of the nanoporous membrane and improve stability and
life span of the biosensor of the present invention.

[0077] The conductive hydrogel can also include a conductive polymer. Any
suitable conductive polymer matrix is suitable in the biosensor of the
present invention. For example, the conductive polymer can include a
polymer of poly(3,4-ethylenedioxythiophene) (PEDOT). Other conductive
polymer are also useful in the present invention, including, but not
limited to, polypyrroles, polythiophenes, etc.

[0078] The conductive hydrogel can be prepared by any suitable means. For
example, the PEG hydrogel can be prepared first using vinyl
polymerization methods, followed by preparation of the PEDOT polymer
using electropolymerization methods. The vinyl polymerization methods
include acid catalyzed or radical initiated polymerization. In some
embodiments, polymerization of the PEG can be photoinitiated. The PEDOT
polymer can be a copolymer of several EDOT monomer units, including EDOT
and EDOT-COOH. When the EDOT monomers are copolymerized, a PEDOT random
copolymer can be formed. The PEDOT copolymer can be any suitable
molecular weight, with any suitable distribution of EDOT and EDOT-COOH
comonomers. For example, the ratio of EDOT and EDOT-COOH comonomers can
be from about 1000:1 to about 1:1000, or about 500:1, 100:1, 50:1, 10:1,
9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,
1:7, 1:8, 1:9, 1:10, 1:50, 1:100 or about 1:1000. Other ratios of the
monomers are also useful.

[0079] In some embodiments, the PEDOT random copolymer can have the
structure according to Formula I:

##STR00008##

wherein each R is independently selected from the group consisting of
--OH and the biorecognition element, wherein at least one R is the
biorecognition element, and x and y are independently an integer of from
about 1 to about 1000, wherein the sum of x and y is an integer of from
about 2 to about 1000. In some embodiments, the ratio of x to y is from
about 10:1 to about 1:10. In some embodiments, the ratio of x to y is
about 1:4.

[0080] The PEDOT random copolymer can be prepared before or after
preparation of the PEG hydrogel. In some embodiments, the PEDOT random
copolymer is prepared after preparation of the PEG hydrogel by depositing
the EDOT and EDOT-COOH monomers in the PEG hydrogel and exposing the PEG
hydrogel to electropolymerization conductions such that the PEDOT random
copolymer is prepared in the PEG hydrogel, but is not covalently bound to
the PEG hydrogel.

[0081] The present invention includes any suitable biorecognition element.
For example, the biorecognition element can include an antibody, an
aptamer, a camelid, a peptide, a protein, or other biological agent. In
some embodiments, the biorecognition element can be a peptide, an
antibody, an enzyme or an aptamer. In some embodiments, the
biorecognition element can be an antibody. In some embodiments, the
biorecognition element can be an enzyme. In some embodiments, the
biorecognition element can be an aptamer. In some embodiments, the
biorecognition element can be IFN-.gamma.. In some embodiments, the
biorecognition element can be bovine-IFN-.gamma..

[0082] Any suitable aptamer or enzyme can be used as a biorecognition
element in the present invention. Aptamers in the present invention may
be specific to inflammatory cytokines including but not limited to
TNF-.alpha., IL-2, IL-4, IL-6, IL-10, IL-17, IL-21 and TGF-.beta..
Aptamers may also be specific to cell surface markers including but not
limited to CD4, CD8, CD36, CD 14, CD45 and EpCAM. Aptamers may also be
specific to components of pathogens including but not limited to HIV,
HBV, HCV and sexually transmitted diseases (STDs). Aptamers may also be
specific to transmembrane proteins present in extracellular vesicles for
example CD63. Any redox enzyme may be used as a biorecognition element in
the present invention. Major groups of enzymes include but are not
limited to oxidoreductases (e.g. peroxidase, glucose oxidase, lactate
oxidase) and NADH-dependent enzymes.

[0083] Other biorecognition elements include peptides. The peptides can be
any suitable peptide that bind to the disease marker, or serve as the
substrate for proteases. Such a peptide allows the biosensor of the
present invention to detect protease activity.

[0084] The biorecognition element can be linked to the conductive polymer,
or directly to the electrodes. In some embodiments, the biorecognition
element can be linked to the conductive polymer. When the conductive
polymer is the PEDOT random copolymer, the biorecognition element can be
linked to the carboxylic acid group of the PEDOT random copolymer. In
some embodiments, the biorecognition element is covalently bound to the
PEDOT random copolymer embedded within the nanoporous membrane, wherein
the nanoporous membrane comprises a PEG hydrogel having PEG chains with a
molecular weight of from about 1000 Da to about 10,000 Da.

[0085] In some embodiments, the biorecognition element can be covalently
linked to the electrodes. When the biorecognition element is covalently
linked to the electrodes, the electrodes can be modified with a
self-assembled monolayer capable of covalently linking to the
biorecognition element. In some embodiments, the biorecognition element
is covalently linked to the electrode to form a self-assembled monolayer
of the biorecognition element on the electrode. In some embodiments, the
biorecognition element is an aptamer having a redox reporting moiety and
a thiol moiety, wherein the thiol moiety is covalently bound to a gold
working electrode.

[0086] The biosensor of the present invention can have one biorecognition
element or several different biorecognition elements. For example, the
PEDOT conductive polymer can be functionalized with several different
biorecognition elements. This allows for the simultaneous detection of
several different disease markers, as well as greater flexibility when
testing for one of several different disease markers. Similarly, when the
biorecognition element is linked directly to the electrode, each
electrode can be modified with one biorecognition element or several
different biorecognition elements. See, for example, Liu et al.,
"Detecting multiple cell-secreted cytokines from the same
aptamer-functionalized electrode" Biosensors and Bioelectronics 2015, 64,
43-50, incorporated herein in its entirety.

[0087] The biosensor of the present invention can include a variety of
other components. For example, the biosensor can include a microtiter
well plate, a well cover, among others. In some embodiments, the
biosensor also includes a microtiter well plate for housing the
biosensor. In some embodiments, the biosensor also includes a well cover
comprising one or more walls contacting the substrate, the walls defining
a well perimeter surrounding the electrode.

IV. Methods for Detecting Disease Markers

[0088] The present invention also provides methods for using the biosensor
of the present invention for disease detection by detecting disease
markers. In some embodiments, the present invention provides a method for
detecting a disease marker in a biological sample comprising contacting a
sensor of the present invention with the biological sample and detecting
the binding of the disease marker to a biorecognition element, thereby
detecting the disease marker.

[0089] The detecting can be performed by any suitable means or device. For
example, the detecting can involve detecting a change in the electrical
signal generated by the biosensor of the present invention. Without being
bound by theory, when the disease marker binds to the biorecognition
element, a change in the electrical signal of the nanoporous membrane is
generated and transmitted from the biorecognition element to the
electrode via the conductive polymer.

[0090] In some embodiments, the detecting the binding of the disease
marker to the biorecognition element comprises measuring the peak
reduction current of the PEDOT random copolymer. In some embodiments, the
binding of the disease marker to the biorecognition element is detected
using square wave voltammetry.

[0091] Any suitable disease marker can be used for detection. For example,
the disease marker can be a peptide, protein, antibody, enzyme, cell,
tissue, etc. from the patient. In some embodiments, the disease marker
can be interferon. In some embodiments, the disease marker can be
interferon-gamma. Other disease markers include exosomes, extracellular
vesicles (EVs). The extracellular vesicles contain transmembrane proteins
that can be detected using specific aptamers. Exemplary transmembrane
proteins include, but are not limited to, CD63. Other disease markers
include proteases such as, but not limited to, matrix metalloproteinases
(MMPs) MMP2, MMP4, and MMP9, as well as urokinase (uPA).

[0093] In some embodiments, the disease marker is indicative of an
infection by tuberculosis or hepatitis C. In some embodiments, the
disease marker comprises IFN-.gamma.. In some embodiments, the
biorecognition element comprises an interferon-gamma (IFN-.gamma.)
antibody. In some embodiments, the biorecognition element comprises an
aptamer specific for IFN-.gamma..

V. Conductive Hydrogels

[0094] The present invention also provides conductive hydrogels for use in
the biosensors of the present invention. In some embodiments, the present
invention provides a conductive hydrogel including a covalently
cross-linked poly(ethylene glycol) (PEG) hydrogel; and a
poly(3,4-ethylenedioxythiophene) (PEDOT) random copolymer embedded within
the PEG hydrogel, the PEDOT random copolymer having a structure according
to Formula I:

##STR00009##

wherein each R is independently selected from the group consisting of
--OH and a biorecognition element selected from the group consisting of a
peptide, an antibody, an enzyme, and an aptamer, wherein at least one R
is the biorecognition element, and x and y are independently an integer
of from about 1 to about 1000, wherein the sum of x and y is an integer
of from about 2 to about 1000.

[0095] In some embodiments, the PEG hydrogel comprises PEG chains having
molecular weights of from about 1000 Da to about 10,000 Da. In some
embodiments, the PEG hydrogel comprises PEG chains having a molecular
weight of about 6000 Da. In some embodiments, the ratio of x to y is from
about 10:1 to about 1:10. In some embodiments, the ratio of x to y is
about 1:4. In some embodiments, the covalently cross-linked poly(ethylene
glycol) hydrogel is prepared from PEG-diacrylate.

VI. Methods for Preparing Conductive Hydrogel Biosensors

[0096] The conductive hydrogels of the present invention can be prepared
by any means known to one of skill in the art. For example, a PEG
hydrogel can be prepared first, and then a conductive polymer, such as
PEDOT, can be polymerized within the PEG hydrogel so that the conductive
polymer is embedded in the PEG hydrogel but not covalently linked to the
PEG hydrogel.

[0097] In some embodiments, the present invention provides a method for
preparing the conductive hydrogel of the present invention, including
contacting a PEG-diacrylate and a photoinitiator under polymerization
conditions suitable to form a PEG hydrogel; contacting the PEG hydrogel
with a solution comprising 3,4-ethylenedioxythiophene (EDOT) and
2,3-dihydrothieno[3,4-b][1,4]dioxine-2-carboxylic acid (EDOT-COOH) under
electropolymerization conditions sufficient to form a
poly(3,4-ethylenedioxythiophene) (PEDOT) random copolymer of Formula I
embedded within the PEG hydrogel:

##STR00010##

wherein each R is --OH, and x and y are each an integer of from about 1
to about 1000, wherein the sum of x and y is an integer of from about 2
to about 1000; and contacting the hydrogel with a biorecognition element
under conditions sufficient to covalently bind the biorecognition element
to the PEDOT random copolymer, thereby forming the PEDOT random copolymer
of Formula I wherein at least one R is a biorecognition element selected
from the group consisting of a peptide, an antibody, and an aptamer,
thereby preparing the conductive hydrogel of the present invention.

[0098] The PEG-diacrylate can be any suitable PEG-diacrylate having the
formula:

##STR00011##

The PEG portion of the PEG-diacrylate can have any suitable molecular
weight, as described above. In some embodiments, the PEG-diacrylate has a
molecular weight of from about 1000 Da to about 10,000 Da. In some
embodiments, the PEG-diacrylate has a molecular weight of about 6000 Da.

[0099] The PEG hydrogel can be prepared by any means known to one of skill
in the art. For example, the PEG can be functionalized at one or both
ends with a suitable polymerizable group. Suitable polymerizable groups
include, but are not limited to, acrylates, methacrylates, styrenics,
cyanoacrylates, and others. The polymerization of the PEG hydrogel can be
performed under radical polymerization conditions, or acid or base
polymerization conditions. In some embodiments, the polymerization is
performed under radical polymerization conditions. In some embodiments,
the polymerization conditions for forming the PEG hydrogel comprises
irradiating the PEG-diacrylate.

[0100] When the PEG hydrogel is prepared by irradiating the PEG-diacrylate
with a photoinitiator, the irradiation can be performed under a variety
of conditions. For example, the PEG hydrogel can be irradiated for from 1
second to more than 1 minute, including 2, 3, 4, 5, 10, 15, 20, 30, 40,
and 50 seconds. Irradiation for longer periods is also contemplated by
the present invention. The wavelength and intensify of irradiation can
depend on the photoinitiator used, the concentration of photoinitiator,
and the degree of crosslinking desired in the PEG hydrogel.

[0101] The photoinitiator can be any suitable photoinitiator known in the
art. For example, the photoinitiator can be azobisisobutyronitrile,
benzoyl peroxide, camphorquinone, or
1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one
(IRGACURE.RTM. 2959 from Ciba).

[0102] The conductive polymer can be polymerized by any means known to one
of skill in the art, such as electropolymerization. As discussed above,
the conductive polymer can be a single polymer or a copolymer.
Representative polymers including polythiophenes, polypyrroles, and
polyphenylenes. When the conductive polymer is a polythiophene based
polymer, any suitable thiophene-based monomer can be used. Representative
thiophene monomers include, but are not limited to, thiophene,
3-methylthiophene, 3,4-ethylenedioxythiophene (EDOT), and
2,3-dihydrothieno[3,4-b][1,4]dioxine-2-carboxylic acid (EDOT-COOH). The
EDOT-COOH monomer includes a carboxylic acid functional group that can be
functionalized with the biorecognition element.

[0103] In some embodiments, the conductive polymer is a polythiophene
polymer. In some embodiments, the conductive polymer is a PEDOT
copolymer. When the conductive polymer is a PEDOT copolymer, the EDOT and
EDOT-COOH monomers can be present in any suitable ratio, as described
above. In some embodiments, the EDOT and EDOT-COOH are present in a ratio
of from about 10:1 to about 1:10. In some embodiments, the EDOT and
EDOT-COOH are present in a ratio of about 1:4.

[0104] The conductive polymer can be modified with the biorecognition
element using any methods known to one of skill in the art. For example,
when the conductive polymer is a PEDOT copolymer using EDOT-COOH, the
biorecognition element can be linked to the PEDOT copolymer using amide
formation chemistry. Preparation of activated esters, such as via EDC
chemistry, are well known and useful in the present invention.

[0105] The biorecognition element can be any suitable agent as described
above. In some embodiments, biorecognition element is an antibody. In
some embodiments, the biorecognition element is an aptamer.

VII. Examples

Example 1

Preparation of PEDOT-PEG Hydrogel

[0106] We constructed a novel composite conducting hydrogel constituting
conductive carboxyl-functionalized PEDOT and a high molecular weight PEG
for development of an immunosensor. The carboxyl functionality provides
capability of bioconjugation. The bio-interfaces were prepared by
polymerizing PEG on top of miniaturized Au electrodes followed by
electro-polymerizing PEDOT copolymer into the porous PEG gel using
aqueous microemulsion. The PEDOT/PEG copolymer was compositionally
tunable and controlled to deposit on an array of electrode surfaces. The
COOH groups present on the conducting polymer chain were tailored with
B-IFN-.gamma. antibodies via active ester groups. The antibody
immobilized conducting hydrogel electrodes were utilized for label-free
electrochemical detection of B-IFN-.gamma. in buffer and bovine plasma.
Binding of B-IFN-.gamma. to antibody results in electrochemical signal
decrease caused due hindrance of electron transfer. This signal-off
sensing method does not need any external redox labels for detection.

[0107] The process of fabricating sensing surfaces begins by
photopolymerizing PEG hydrogel on top of electrodes. Subsequently, PEDOT
copolymer is electropolymerized within PEG hydrogel. The electrodes
deposited with conductive hydrogel showed faint blue when it is oxidized
and turned dark blue when it is reduced due to its intrinsic properties
(FIG. 7A). Scanning electron microscopy (SEM) was used to investigate the
morphological features of conducting polymer and conducting hydrogel
surfaces. As seen from SEM images (FIG. 7B), the PEDOT deposition on a
gold electrode showed rough granular morphology and UV polymerized PEG
hydrogel showed porous morphology. The electropolymerization of PEDOT
inside PEG fills the pores in PEG. Sole PEDOT polymerization on Au
surfaces suffered from poor mechanical performances.

[0108] Importantly, PEDOT molecules within the gel undergo redox reactions
upon application of voltage and possess characteristic redox peaks. The
cyclic voltammograms of PEG hydrogel without and with incorporation of
conductive PEDOT revealed enhanced electrical properties of conductive
hydrogel over the regular PEG hydrogel (FIG. 10A). The incorporation of
conducting polymer in PEG showed greater redox current values by almost
100 fold. The sharp intrinsic oxidation and reduction property of PEDOT
was further utilized for the biosensing application. PEDOT/PEG hydrogel
showed similar CV characteristic to PEDOT which proves that PEG did not
affect the electrochemical behavior of PEDOT (FIG. 10B).

[0109] The deposition of PEDOT inside the PEG gel was optimized by varying
the applied charges for the electropolymerization. As seen from the data
of redox behavior as a function of charge deposited, we could confirm
that the enhanced redox properties at the charge of 8.times.10.sup.-4 C,
while the lower and higher charge (or thickness of PEDOT film) leads to
poor redox peaks and reduction current (FIG. 13A-13D).

[0110] A pre-polymer PEG hydrogel solution, 5% PEG-diacrylate and 1%
photoinitiator (Irgacure 2959) in PBS was coated onto the Au electrodes
of the patterned slides. These slides were exposed to UV radiation (60
mJ/cm.sup.2) for 5 s through an aligned photo mask on top of the
electrodes. The prepared PEG hydrogel on top of patterned Au electrodes
was then incubated in the aqueous solution of 10 mM of EDOT-COOH
containing 2.5 mM EDOT, 0.05 M SDS and 0.1 M LiClO.sub.4 in the batch
mode setup for electropolymerization. This monomer solution was
electropolymerized into the porous PEG hydrogel amperometrically at a
constant potential of 1.1 V using CHI 6044d Electrochemical Analyzer (CH
Instruments, Inc. Austin, Tex.). The resulting polymer has enhanced redox
properties at the charge of 8.times.10.sup.-4 C of the polymer film.

[0111] Antibody is attached covalently via carboxylic groups present on
PEDOT. Carboxylic group was activated to immobilize antibody covalently.
The conducting polymer hydrogel array was incubated with 0.2 mM EDC and
0.05 mM NHS in DI water for 15 min. The array was rinsed with PBS and
incubated with 50 .mu.g/mL bovine INF-.gamma. antibody in PBS overnight
at 4.degree. C. The electrochemical measurement was performed on
conducting polymer hydrogel array using CHI instrument. Cyclic
voltammetry was employed in the range between -0.7V and 0.4V to measure
binding of bovine INF-.gamma.. Peak current (I.sub.p) was measured at
-0.35V during each measurement and the signals were plotted by a function
of concentration versus current values.

[0112] The COOH groups present on the conducting polymer are activated by
standard EDC-NHS chemistry in order to immobilize B-IFN-.gamma. antibody
molecules. A mixture of EDOT-COOH and EDOT in a LiClO.sub.4 aqueous
solution containing sodium dodecyl sulfate (SDS) was polymerized
amperometrically within PEG hydrogel layer at potential 1.1 V keeping a
constant charge of 8.times.10.sup.-4 C. This process yielded an
interconnected polymer structure inside the PEG hydrogel. The number of
COOH groups were quantified by toluidine blue O (TBO)
staining..sup.[24-25] The calibration curve for COOH groups TBO
absorption at 633 nm was obtained (FIG. 14A-14B) the density of the COOH
groups was estimated to be 3.6.times.10.sup.16 molecules/cm.sup.2 which
was ca. 2-fold higher than the density of COOH found in electrochemically
deposited PEDOT-COOH/PEDOT on bare gold surface (1.7.times.10.sup.16
molecules/cm.sup.2).

[0113] Anti-bovine IFN-.gamma. antibody was immobilized covalently on
carboxylated PEDOT chains inside the conducting polymer hydrogel using
EDC-NHS. Response of antibody immobilized conducting polymer hydrogel was
studied in PBS using cyclic voltammetry. This highly conducting hydrogel
matrix gives response even in PBS and obviates the need of external redox
indicators. The antibody immobilized conducting polymer hydrogel surface
was challenged with different concentrations of recombinant B-IFN-.gamma.
and the cyclic voltammograms were recorded. Capture of B-IFN-.gamma. by
antibody-containing gel hinders electron transfer through the polymer
chain and causes the redox peak to decrease (FIG. 11C). The drop in the
PEDOT reduction peak in response to different concentration of
B-IFN-.gamma. forms the basis of electrochemical sensing of this analyte.

[0114] We obtained a calibration curve for recombinant B-IFN-.gamma.
concentration versus reduction current of antibody functionalized
conducting hydrogel on electrodes (FIG. 11D). We believe that the binding
of electrochemically insulating target molecule to the antibody
functionalized PEDOT chains hinders the charge transfer properties and
conductivity of PEDOT that results in suppression of reduction current.
Additionally, sandwich assays were performed on ITO electrodes to
visualize the immobilized B-IFN-.gamma.. The electrodes were then
incubated with recombinant B-IFN-.gamma. followed by biotinylated
secondary antibody and fluorophore-conjugated streptavidin. The
fluorescence was measured using a fluorescent microscope. Incubation with
fluorescently labeled antibody revealed the binding of recombinant
B-IFN-.gamma. to the antibody immobilized conductive hydrogel (FIG. 15A:
fluorescence images).

Example 2

Preparation of Sensor

[0115] Gold and ITO patterned electrodes were prepared using
photolithography and wet-etching approaches as previously reported. The
micropatterned glass slides, containing photoresist on top of Au surface,
were treated with oxygen plasma for 10 min and incubated in 0.05%
solution of (3-acryloxypropyl) trichlorosilane in toluene for about one
hour under nitrogen atmosphere to obtain a self-assembled monolayer of
silane on the glass regions. The procedure is described in more detail
below.

[0116] Positive resist (S1813) was spin-coated (2000 rpm for 30 sec) on
glass slides coated with 15 nm Cr adhesion layer and 100 nm Au layer
resulting in formation of a thick layer of photoresist. The
photoresist-coated glass slides were soft-baked on a hot-plate at
115.degree. C. for 1 min, then placed in contact with a photomask and
exposed to 365 nm UV source. The substrates were then placed into a
developer solution (MF 319). After development step, Au-coated glass
slides were immersed in Au etching solution followed by immersion in Cr
etching solution. Metal was selectively removed from the regions not
protected by photoresist, resulting in formation of Au micropatterns of
diameter 1500 am. Importantly, the photoresist layer on top of Au
microelectrodes was not removed immediately after etching but was
employed to protect underlying Au regions during the silane modification
protocol so as not to lose conductivity.

[0117] The substrates with photoresist-covered Au electrodes were modified
with 3-(acryloxypropyl) tricholosilane. After silane modification,
substrates were sonicated in acetone to remove photoresist.

[0118] The nanoporous membrane including the PEDOT-PEG hydrogel described
in Example 1 is then prepared on top of the electrodes and becomes
attached to the substrate. The biorecognition element is then attached to
the PEDOT-PEG hydrogel, again, following the procedure in Example 1.

Example 3

Detection of Tuberculosis

[0119] The antibody (bovine-IFN-.gamma.) functionalized PEDOT conducting
hydrogel was placed in the electrochemical cell set up of Example 2.
Different concentrations of target (recombinant interferon gamma)
prepared in 1.times.PBS were introduced into the electrochemical cell and
incubated for about 20 mins. The cyclic voltamograms were recorded.

[0120] Each sensing electrode (antibody functionalized conductive hydrogel
on Au microelectrode) was connected to potentiostat via Au contact pads
as shown in FIG. 8A. Each sensing electrodes was individually addressed
by potentiostat in this way.

[0121] The Au electrodes having conductive gel on the sensing chip were
working electrodes and were individually addressed by potentiaostat. We
used external Ag/AgCl reference and platinum wire counter electrodes.
These were dipped in the lectrolyte solution of the electrochemical cell
set up as shown in Fig. and were connected to potentiostat.

[0122] To test the specificity of the conducting hydrogel bioelectrodes,
the sensor was challenged with nonspecific cytokines and proteins
including TGF-.beta., IL-6, TNF-.alpha., and IgG. We observed minimal
signal reduction with the nonspecific cytokines (FIG. 11A) and human
IFN-.gamma. (FIG. 11B). This result demonstrates that our sensor can
detect B-IFN-.gamma. in a mixture of nonspecific cytokines. To
demonstrate feasibility of our sensor to cell-related experiments, we
wanted to detect signal responses in serum-containing media and whole
blood. As shown in FIG. 11C, addition of whole blood resulted in
negligible signal loss as compared to RPMI media alone. The antibody
immobilized conductive hydrogel sensor was challenged with the known
B-IFN-.gamma. spiked in (1:1) TB-free bovine blood/PBS (FIG. 16A-16B).
The addition of bovine blood resulted in signal loss of .about.20%
compared to PBS, however, despite this loss in signal, changes were
detected upon adding serial concentrations of B-IFN-.gamma.. Using bovine
plasma samples purified from bovine blood samples, we compared the
results from our conducting polymer hydrogel to ELISA method and found
correlation between ELISA results and electrochemical signals (FIG. 11D).
For real time detection of B-IFN-.gamma. release from bovine blood cells,
the antibody immobilized conductive hydrogel was challenged with bovine
peripheral blood mononuclear cells (PBMCs). Upon mitogenic stimulation,
the change in electrochemical current was observed (FIG. 11E). As
controls, the current did not change on the antibody-absent conductive
hydrogel with stimulated cells or antibody-immobilized conductive
hydrogel with non-stimulated cells. Thus, in the present study, we could
confirm our sensor was specific and responsive for B-IFN-.gamma..

[0123] Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, one of skill in the art will appreciate that certain
changes and modifications may be practiced within the scope of the
appended claims. In addition, each reference provided herein is
incorporated by reference in its entirety to the same extent as if each
reference was individually incorporated by reference. Where a conflict
exists between the instant application and a reference provided herein,
the instant application shall dominate.